Methyl-binding domain protein-based DNA isolation from human blood serum combines DNA analyses and serum-autoantibody testing

Abstract

Background: Circulating cell free DNA in serum as well as serum-autoantibodies and the serum proteome have great potential to contribute to early cancer diagnostics via non invasive blood tests. However, most DNA preparation protocols destroy the protein fraction and therefore do not allow subsequent protein analyses. In this study a novel approach based on methyl binding domain protein (MBD) is described to overcome the technical difficulties of combining DNA and protein analysis out of one single serum sample.

Methods: Serum or plasma samples from 98 control individuals and 54 breast cancer patients were evaluated upon silica membrane- or MBD affinity-based DNA isolation via qPCR targeting potential DNA methylation markers as well as by protein-microarrays for tumor-autoantibody testing.

Results: In control individuals, an average DNA level of 22.8 ± 25.7 ng/ml was detected applying the silica membrane based protocol and 8.5 ± 7.5 ng/ml using the MBD-approach, both values strongly dependent on the serum sample preparation methods used. In contrast to malignant and benign tumor serum samples, cell free DNA concentrations were significantly elevated in sera of metastasizing breast cancer patients. Technical evaluation revealed that serum upon MBD-based DNA isolation is suitable for protein-array analyses when data are consistent to untreated serum samples.

Conclusion: MBD affinity purification allows DNA isolations under native conditions retaining the protein function, thus for example enabling combined analyses of DNA methylation and autoantigene-profiles from the same serum sample and thereby improving minimal invasive diagnostics.

Figure 1

Extraction of cell free DNA from control individuals. Box plot of DNA amount isolated from 1 ml serum or plasma from (1) Austrian Institute of Technology (n = 12), (2) Austrian Red Cross (n = 24) and (3) AKH (n = 24). Serum DNA levels were dependent on serum source. Using the silica-based extraction protocol, mean amounts of DNA could be isolated ranging from (1), 11.9 ± 10.9 ng/ml (mean ± SD) and (3), 12.2 ± 9.7 ng/ml to (2), 39.7 ± 32.8 ng/ml. By contrast using the MBD-based protocol, serum DNA concentrations of (1) 2.5 ± 1.9 ng/ml, (2) 11.5 ± 7.3 ng/ml and (3) 2.4 ± 1 ng/ml were observed.

Figure 2

Increased level of cell free DNA in serum of Breast cancer patients with metastasizing disease. Amount of cell free DNA isolated from 1 ml serum of breast cancer patients with metastasizing tumors and control individuals obtained from AKH (source 3). An increased serum DNA amount was detected in sera from metastasizing tumors with both isolation strategies. (A), silica based isolation protocol (P = 0.0043, Wilcoxon test); (B) MBD loaded bead based purification (P = 0.0021, Wilcoxon test).

Figure 3

Qualitiy of cell free serum DNA. (A) reflects the amplification success for each sample using MBD or silica membrane based serum DNA. A maximum of 12 fragments per sample was possible and in sum 34 samples per isolation approach were analyzed. (B) shows the amplification success of each fragment getting amplified across all analyzed samples. Both plots (A, B) are based on the analysis of two multiplex PCRs performed on serum or plasma DNA isolates (source 1, 2).

Figure 4

Autoantibody tests of MBD processed serum. Pearson correlation plots upon X-values of autoantibody protein micro arrays analyzing serum and plasma samples with and without MBD treatment. All samples originate from source 1 (AIT). Comparison of plasma and serum samples were performed on samples obtained from one single blood withdrawal. The cor-value states the Pearson's correlation.